Nisin as an antibotulinal agent for food products

ABSTRACT

Processed foods and food products are prepared by including from 2,000 to 10,000 I.U. of nisin to prevent the outgrowth of Clostridium botulinum spores.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of my earlier applicationSer. No. 543,968 filed Oct. 20, 1983, which in turn is a continuation ofmy earlier application Ser. No. 503,305 filed June 10, 1983 nowabandoned.

BACKGROUND OF THE INVENTION

Nisin, an antimicrobial substance produced by certain strains ofStreptococcus lactis, has known food preservative use. It has theproperty of inhibiting the growth of certain Gram-positive bacteria, butnot Gram-negative ones or yeasts or molds. A further, and most importantpractical property, lies in the ability of nisin to prevent theoutgrowth of germinate bacterial spores. The commercial use of nisin todate very much relates to the property of preventing the outgrowth ofgerminated bacterial spores, in some applications that involves theprevention of spoilage in foods which have received at least sufficientheat treatment to destroy vegetative bacterial cells. Another classicuse has been for the prevention of clostridial spoilage in processcheese products.

The use of nisin in process cheese products has been the subject ofearlier patents; see British Pat. No. 713,251 and U.S. Pat. No.2,744,827 dated 1954 and 1956, respectively. These patents relate to themanufacture or preservation of cheese of kinds liable to become blown ordistended due to the growth of spoilage organisms which are anaerobicsporeformers, typically bacteria known as Clostridia. They also relateto process cheese which is liable to be spoiled as above. The levels ofnisin referred to in these patents are in the range of 50 to 375 unitsper gram. One "unit" is equivalent to 1 International Unit (I.U.) ofNisin activity and is equivalent to 0.025 microgram of Nisin.

Until recent times it was not considered that process cheese productswould be liable to spoilage by Clostridium botulinum organisms or thatthe production of the associated toxin would present a potential healthrisk. Recent findings however are that such risks can exist,particularly in process cheese formulations where the water content isin excess of 54 percent. See Kautter et al "Toxin Production byClostridium botulinum in Shelf-Stable Pasteurized Process CheeseSpreads". Journal of Food Protection, 42 pp. 784-786 (1979); Tanaka etal. "A Challenge of Pasteurized Process Cheese Spreads with Clostridiumbotulinum spores", Journal of Food Protection, 42 pp. 787-789 (1979);Scott and Taylor, "Effect of Nisin on the Outgrowth of Clostridiumbotulinum Spores", J. Food Sci., 46(1) pp. 117-120 (1981); Scott andTaylor, "Temperature, pH, and Spore Load Effects on the Ability of Nisinto Prevent on the Outgrowth of Clostridium botulinum Spores", J. FoodSci. 46(1): 121- 126 (1981); and Somers and Taylor, ResearchNote--"Further Studies on the Antibotulinal Effectiveness of Nisin inAcidic Media", J. Food Sci., 46(6): p. 1972-3.

Our studies at the fundamental level have shown that nisin does have theability to inhibit growth of botulinum spores from different culture andtype sources. The significant finding, however, is that the levels ofnisin required to effect complete inhibition are substantially higherfor Clostridium botulinum than for other non-pathogenic clostridia oraerobic sporeformers commonly encountered as food spoilage organisms. Wehave further demonstrated in a protracted shelf-life study withhigh-moisture, reduced sodium process cheese spread that completeinhibition of Clostridium botulinum spore outgrowth is achieved at anisin addition level of 250 ppm (10,000 IU nisin/gram). This nisin levelis 20 times the maximum addition rate in current commercial practice.

A demand is present for a reliable antibotulinal agent to be used infoods and food systems susceptible to C. botulinum growth either tosupplement current used materials such a nitrate and sorbate or as thesole antibotulinal agent used in such foods.

BRIEF DESCRIPTION OF THE INVENTION

The present invention includes a process for substantially completelyinhibiting the outgrowth of germinated Clostridium botulinum spores infood products. Included herein is the use of nisin in high moisturecontent pasteurized process cheese spreads, typically having a moisturecontent in the range of about 54 to about 60% by weight and havingreduced sodium levels in the range of 40 to about 50% reductions inadded sodium salts from the typical commercial formulations (whichcontain 2.55% disodium phosphate and 2.0% sodium chloride), comprisingtreating this process cheese by adding to it an amount of nisin ornisin-producing bacterial culture sufficient to inhibit botulinum sporeoutgrowth, the amount generally being from about 2,000 to about 10,000International Units (or equivalent). This is the subject of my earlierapplications Ser. Nos. 503,305 and 543,968, identified above, thedisclosures of which are hereby incorporated by reference.

The use of nisin to control heat-resistant spores of Clostridumbotulinum may be applied to broad categories of foods and edible foodproducts, especially those products known or determined to besusceptible and/or hospitable to C, Botulinum growth. Low acid foodswith pH values above about 4.5 such as tomato products, cold meatproducts, wet fish systems and the like are examples. The use ofsignificant amounts of nisin sufficient to control the outgrowth of C.botulinum spores and the elaboration of botulinum spores therefrom inthe following foods is specifically contemplated: canned peas and beans,canned mushrooms, canned tomato products, soups and the like. Nisindelays toxin production by C. botulinum type E spores in cod filetsstored at 80° F. and exhibits antibotulinal effects either alone or withnitrite in chicken frankfurter emulsion. The required amount of nisin isconveniently mixed into the food in such a way as to bring it intocontact with any spores which are present.

The quantity of nisin applied necessary to control the growth ofClostridum botulinum spores and to impede or prevent the elaboration ofbotulinum toxin therefrom depends upon numerous factors including thenature of the food itself, pH, spore loading both prior to and afterprocessing, packing conditions and practices, etc. The requisite amountof nisin may be determined by testing procedures in the manner of thosedescribed below. As a guideline the following dosages may be considered:

canned peas and beans: 100-150 mg/kg

canned mushrooms: 100-200 mg/kg

canned soups: 100-200 mg/kg

canned tomato products: 50-100 mg/kg

(pH typically below 4.5)

Toxicological evaluation by the Joint FAO/WHO Expert Committee of FoodAdditives noted in 1968 that evidence then available indicated that alevel of 3,300,000 International Units of nisin per Kg of bodyweight hadno adverse effect. This finding permits an unconditional ADI of 33,000units per Kg body weight at which level nisin has no microbiological,toxic, or allergic effects. Thus an amount of nisin will be used thatachieves the required antibotulinal effect yet is well within acceptedtoxicological limits.

Human botulism is almost invariably the result of eating preserved foodsin which the bacteria, Clostridium botulinum, has grown and producedtoxin. It appears that, for example, as the moisture content ofnon-refrigerated, pasteurized process cheese spreads or productsincreases, so too does the incidence of toxin produced by Clostridiumbotulinum. The species Clostridium botulinum is divided into Types Athrough G on the basis of antigenic differences between the toxins:Types A, B, and E are of practical significance in the United Statesbecause they are the ones that are most commonly involved in humanintoxications. Of these, Type A toxin is the most potent toxin and isreported to be the most potent toxic substance known.

Nisin is a polypeptide produced by some Streptococcus lactis strains oflactic streptococci. The World Health Organization Committee onBiological Standardisation has established an international referencepreparation of nisin, and the international unit (IU herein) is definedas 0.001 mg of this preparation. The most potent Nisin preparationprepared contains 40 million IU per gram. NISAPLIN is a commercial Nisinconcentrate containing 1 million IU per gram commercially available fromAplin & Barrett Ltd., Trowbridge, Wiltshire, England.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

To determine the effectiveness of nisin in preventing the outgrowth ofClostridium botulinum spores and subsequent toxin formation in processcheese spreads, the following experiments were conducted.

Bacterial cultures: Five strains each of Clostridium botulinum types Aand B (A: 56, 62, 69, 77, and 90; B: 53, 113, 213, 13983, andLamanna-okra) were used. Each strain was grown in a manner conducive tosporulation as in Christiansen et al, Appl. Microbiol. 27, pp. 733-737.The spore crops were harvested by centrifugation followed by thoroughwashing with sterile water. The final pellet was resuspended in sterilewater to 10 ml. Each individual spore suspension was thawed and dilutedin 0.1% peptone-water for enumeration of the spores (see below). Afterenumeration, suitable dilutions of the individual spore preparations insterile water were made and combined to produce a mixed inoculumconsisting of approximately equal numbers of each strain. These mixedspore preparations wee suitably diluted in 0.1% peptone-water prior toaddition to the melted cheese.

Enumeration of C. botulinum: Enumeration of C. botulinum was conductedby the 5-tube Most Probable Number (hereinafter MPN) technique usingTPYG as the growth medium according to the FDA BacteriologicalAnalytical Manual for Foods (1976). For spore counts, the sporesuspensions were diluted in 0.1% peptone-water and heat-shocked at 80°C. for 10 min. prior to enumeration. With stored process cheese spreadsamples, similar heat-shocking methods were used. Tubes showingbacterial growth were teted for the presence of toxin by the mouseprotection assay. Only toxin-positive tubes were counted in determiningthe Most Probable Number of C. botulinum. TPYG medium consisted of 5%trypticase peptone, 0.5% bactopeptone, 2% yeast extract, 0.4% glucose,and 0.1% sodium thioglycollate. The pH of TPYG medium was adjusted to 6prior to autoclaving. The pH of The peptone-water was not adjusted priorto autoclaving.

Extraction and assay of Toxin: The procedure of Tanaka et al, notedabove, was used for the extraction and assay of the toxin. Ten grams ofeach test sample were mixed with an equal volume of gelatin-phosphatebuffer, pH 6.2. The mixture was centrifuged at 5000×g for 10 min and theaqueous supernatant fraction was analyzed for the presence of toxin. Theextraction procedure allowed complete recovery of added toxin. For toxinassays of TPYG broth employed in the MPN procedure, the medium wascentrifuged at 5000×g for 10 min, and the supernatant was analyzed forthe presence of toxin.

In the mouse protection assay for botulinal toxin (FDA BacteriologicalAnalytical Manual for Foods, 1976), each of 2 mice was inoculatedintraperitoneally with 0.5 ml of the extract from the test sample. Micewere held for up to 4 days and examined for symtoms and deathcharacteristic of C. botulinum intoxication. When death occurred, 2additional mice were challenged with a sample-antitoxin mixture whichwas preincubated at 37° C. for 30 min. Unprotected controls were againinoculated so that they could be tested simultaneously with theprotected mice. Nonspecific deaths due to the cheese extract did notoccur.

Cheese: A blend of American cheese, nonfat dry milk, and whey solids wasemployed. This was blended, a uniform product was sealed inmoisture-proof plastic bags and held frozen until needed. This method ofpreparation insured a uniform source of raw material for allexperiments. The blend was prepared with the lowest possible moisturecontent. The amount of nisin naturally present in this product wasdetermined to be below the lower limit of detectability of the nisinbioassay according to Tramer et al, J. Sci. Food Agric., 15:522-528(1964).

Formulations: Five batches of process cheese spread were prepared. Batch#1 was made to 54% moisture with 1.35% sodium phosphate, 1.1% addedsodium chloride, and 0.2% lactic acid. This batch served as a lowspoilage control. Batches 2 through 5 were prepared at 58% moisture with1.45% sodium phosphate and 1.2% added sodium chloride. Batch 2 containedno nisin; Batch 3 had 12.5 ppm nisin; Batch 4 contained 100 ppm nisin;and Batch 5 contained 250 ppm nisin. The formulations of the variousbatches are tabulated in Table 1.

                  TABLE 1                                                         ______________________________________                                        Formulation of Process Cheese Spreads                                         Batch  Percent  % Sodium   % Added Sodium                                                                           ppm                                     No.    Moisture Phosphate  Chloride   Nisin                                   ______________________________________                                        1      54       1.35       1.1        0                                       2      58       1.45       1.2        0                                       3      58       1.45       1.2        12.5                                    4      58       1.45       1.2        100                                     5      58       1.45       1.2        250                                     ______________________________________                                    

Preparation and inoculation of cheese spreads: The cheese was processedin an agitated, covered, steam-jacketed kettle. The kettle accommodatedthe 3 lb. batches. Each batch was completed by making 4 sub-batches.With use of the small sub-batches, very careful control of moisture wasobtained, and the sub-batches were very similar in composition.

The cheese blend, water, disodium phosphate, sodium chloride, lacticacid, and NISAPLIN, a commercial formulation with 1×10⁶ i.u./g, 74.7%sodium chloride, and 23% nonfat dry milk solids, were weighed into thekettle. The mixture was heated to 88° C. and held for 2 minutes.Uninoculated samples were then withdrawn for moisture, pH, and saltanalyses (see below). The C. botulinum spore mixture was then added tothe remainder of the product to give a final spore level of 1000spores/g. The temperature of the inoculated product was kept at 88° C.for a further 2 minutes with agitation.

The processed product was poured into small glass vials, approximately21 g per vial, to just below the rim and the screw caps were closedtight. 180 vials were prepared from each batch. The filled vials werecooled in a refrigerator for at least 1 hour, placed in anaerobe jars,and incubated as indicated below.

Incubation and sampling schedule: Samples were incubated in 2 differentways. In the first method, samples were incubated for up to 48 weeks at30° C. In the second method, samples were held at 4° C. for 8 weeksfollowed by incubation at 30° C. for up to 48 weeks.

Each experimental batch was examined for spoilage (gas, uneven colorchange, phase separation) at 0, 4, 8, 12, 16, 20, 24, 36, and 48 weeksand if applicable at 0 and 8 weeks of refrigerated storage. Samples wereroutinely tested for toxin at 0, 4, 8, 12, 16, 24, 36, and 48 weeks andif applicable after 8 weeks of refrigerated storage. In addition,samples that showed obvious signs of spoilage were tested for toxin assoon as spoilage was noticed. For toxin analysis, a minimum of 10samples per batch were tested throughout.

The number of viable C. botulinum organisms were determined by the5-tube MPN method on each batch immediately following the formulation ofthe batch. In addition, MPNs were performed on any samples that did notspoil or become toxic by the 48th week of storage (a maximum of 10 jarsper batch).

Nisin analysis was performed on 10 jars of each batch at 0, 4, 8, 12,16, 24, 36, and 48 weeks and, if applicable, at 0, 4, and 8 weeks ofregrigerated storage.

Chemical analysis of the uninoculated samples: Total moisture and sodiumchloride analyses were performed in triplicate by recommended AOACmethods. Two moisture procedures are given in the AOAC Manual (Assoc.Off. Anal. Chem., 12th ed., 1976) by the 100° C. vacuum oven procedurewas used in this study. The procedure in Standard Methods for theExamination of Dairy Products (Bianco et al, 1972) was used to measurethe pH of the finished product. Nisin analysis was performed by theMicrococcus flavus assay method of Tramer and Fowler, noted above.

Discussion of Results: The effectiveness of nisin in preventing theoutgrowth of C. botulinum spores in process cheese spreads incubated at30° C. for up to 48 weeks is shown in Table 2. A total of 7 toxicsamples were found in Batch 1, the low moisture control, during the 48week period. However in Batch 2, which had 57.0% moisture, all of thesamples became toxic by the 8th week of storage. In Batch 3, which alsohad 57.0% moisture but also contained 12.5 ppm nisin, the majority ofthe samples became toxic by the 16th week of incubation although a fewsamples survived the entire 48-week period. Nisin at a level of 12.5 ppmdelayed outgrowth and toxin production but did not prevent it. Incontrast, very few toxic samples were obtained in Batches 4 and 5 duringthe 48-week incubation period. Analysis of samples from Batch 4, whichhad 56.8% moisture and 100 ppm nisin, yielded only 3 toxic samplesduring the entire period. No toxic samples were found during incubationof Batch 5, which had 56.7% moisture and 250 ppm nisin. The MPN data inTable 2 indicates that the spores remained viable during the incubationperiod but that their outgrowth was prevented by nisin. On the basis ofthis data it appears that Nisin exerts a sporostatic effect on spores inprocess cheese spreads.

                                      TABLE 2                                     __________________________________________________________________________    Toxin Production in Process Cheese Spreads                                    Incubated at 30° C.                                                                          Number of Toxic Samples/Number of Samples                                     Analyzed Randomly at the Following Weekly                         Initial.sup.b                                                                       Final.sup.c                                                                         Inspection Periods:                                     Batch                                                                             % Moisture                                                                          MPN/g MPN/g 0   4  8   12 16 24 36 48                               __________________________________________________________________________    1   52.4 ± 0.6                                                                       2.92 × 10.sup.3                                                               7.72 × 10.sup.3                                                                0/10                                                                             0/10                                                                             1/10                                                                              2/10                                                                             0/10                                                                             0/10                                                                             2/10                                                                             0/10                                                          +1.sup.d     +2.sup.c                            2   57.0 ± 0.3                                                                       4.95 × 10.sup.3                                                               --    0/4 4/10                                                                             10/10                                                                             -- -- -- -- --                                                         +68.sup.d                                                                        +8.sup.d                                         3   57.0 ± 0.3                                                                       3.05 × 10.sup.3                                                               5.60 × 10.sup.2                                                               0/4 4/10                                                                             7/10                                                                              8/10                                                                             8/10                                                                             -- -- 1/4                                                        +18.sup.d                                                                        +20.sup.d                                                                         +3.sup.d                                                                         +1.sup.d                                  4   56.8 ± 1.0                                                                       2.38 ×  10.sup.3                                                              4.00 × 10.sup.3                                                               0/4 0/10                                                                             1/10                                                                              0/10                                                                             1/10                                                                             0/10                                                                             0/10                                                                             0/10                                                          +1.sup.d                                         5   56.7 ± 0.3                                                                       2.40 × 10.sup.3                                                               4.50 × 10.sup.2                                                               0/4 0/10                                                                             0/10                                                                              0/10                                                                             0/10                                                                             0/10                                                                             0/10                                                                             0/10                             __________________________________________________________________________     .sup.a Mean ± std. deviation of moisture level in the individual           subbatches                                                                    .sup.b Mean of MPNs obtained for individual subbatches                        .sup.c Mean of MPNs obtained for 2 to 5 of the samples remaining at 48        weeks                                                                         .sup.d Additional number of spoiled and toxic samples identified at that      sampling period                                                          

The antibotulinal effectiveness of nisin in process cheese spreads wasnot altered appreciably by incubation of 4° C. for 8 weeks beforeincubation at 30° C. for 48 weeks (Table 3). As before, a limited numberof toxic samples developed in Batch 1, the low moisture control. WithBatches 2 and 3, spoilage and toxin production occurred early in theincubation period. With Batch 3, toxin production was not delayed by12.5 ppm nisin as it seemed to be in the earlier experiment (Table 2).Again, a limited number of toxic samples were obtained with Batch 4, the100 ppm nisin batch, and no toxic samples were found throughout the48-week period at the 250 ppm nisin level, Batch 5.

                                      TABLE 3                                     __________________________________________________________________________    Toxin Production in Process Cheese Spreads                                    Held for 8 Weeks at 4° C. Before Incubation at 30° C.               Initial.sup.b                                                                       Final.sup.c                                                         Batch                                                                             % Moisture                                                                          MPN/g MPN/g -8.sup.d                                                                         0  4   8   12                                                                              16 24 36 48                             __________________________________________________________________________    1   52.4 ± 0.6                                                                       2.92 × 10.sup.3                                                               4.20 × 10.sup.3                                                               0/10                                                                             0/10                                                                             0/10                                                                              2/10                                                                              --                                                                              0/10                                                                             1/10                                                                             1/10                                                                             0/10                                                                 +3    +2                                2   57.0 ± 0.3                                                                       4.95 × 10.sup.3                                                               --    0/10                                                                             0/10                                                                             10/10                                                                             10/10                                                                             --                                                                              -- -- -- --                                                         +54.sup.e                                                                         +5.sup.e                                      3   57.0 ± 0.3                                                                       3.05 × 10.sup.3                                                               1.50 × 10.sup.2                                                               0/10                                                                             0/10                                                                             9/10                                                                              7/10                                                                              3.sup.e                                                                         2.sup.e                                                                          1.sup.e                                                                          -- 1/2                                                        +16.sup.e                                                                         +22.sup.e                                     4   56.8 ± 1.0                                                                       2.38 × 10.sup.3                                                               2.60 × 10.sup.2                                                               0/10                                                                             0/10                                                                             0/10                                                                              3/10                                                                              --                                                                              2/10                                                                             1/10                                                                             0/10                                                                             0/10                           5   56.7 ± 0.3                                                                       2.40 × 10.sup.3                                                               2.50 × 10.sup.2                                                               0/10                                                                             0/10                                                                             0/10                                                                              0/10                                                                              --                                                                              0/10                                                                             0/10                                                                             0/10                                                                             0/10                           __________________________________________________________________________     .sup.a Mean ± std. deviation of moisture levels in the individual          subbatches                                                                    .sup.b Mean of MPN obtained for individual subbatches                         .sup.c Mean of MPNs obtained for 2 to 5 of the samples remaining at 48        weeks                                                                         .sup.d Data obtained at onset of 8week period of refrigerated storage         .sup.e Additional number of spoiled and toxic samples identified at that      sampling period                                                          

Chicken Frankfurter Emulsions: The effectiveness of nisin in preventingtoxin production by Clostridium botulinum in chicken frankfurtheremulsions challenged the 1000 C. botulinium spores per gram wasinvestigated in the following studies.

Bacterial cultures: a 10 strain mixture consisting of five strains eachof Clostridium botulinum types A and B (A: 56, 62, 69, 77 and 90; B: 53,113, 213, 13983, and Lamanna-okra) was used. Each strain was grown in amanner conducive to sporulation. The spore crops were harvested bycentrifugation followed by thorough washing with sterile water. Thefinal pellet was resuspended in sterile water to 10 ml, and theindividual spore suspensions were stored frozen. A portion of eachindividual spore suspension was thawed and diluted in 0.1% peptone-waterfor enumeration of the spores (see below). After enumeration, suitabledilutions of the individual spore preparations in sterile water weremade and combined to produce a mixed inoculum consisting ofapproximately equal numbers of each strain. These mixed sporepreparations were suitably diluted in 0.1% peptone-water prior toaddition to the frankfurter emulsion.

Enumeration of C. Botulinum: C. botulinum was enumerated by the 5-tubeMost Probable Number technique using TPYG as the growth medium. Forspore counts, the spore suspensions were diluted in 0.1% peptone-waterand heat-shocked at 80° C. for 10 min. prior to enumeration. With thestored frankfurter samples, similar heat-shocking methods were used.Tubes showing bacterial growth were tested for the presence of toxin bythe mouse protection assay. Only toxin-positive tubes were counted indetermining the Most Probable Number of C. botulinum.

Media: TPYG medium consisted of 5% trypticase peptone, 0.5%Bacto-peptone, 2% yeast extract, 0.4% glucose, and 0.1% sodiumthiogly-collate. The pH of TPYG medium was adjusted to 7 prior toautoclaving. The peptone-water consisted of 0.1% Bacto-peptone indeionized water. The pH of 0.1% peptone-water was not adjusted prior toautoclaving.

Antitoxin: C. Botulinum anti A/B toxin was obtained from a local source.

Extraction and assay of toxin: The procedure of Tanaka et al was usedfor the extraction and essay of the toxin. Briefly, the sample wasweighed, transferred into a Stomacher bag, and an equal volume ofgel-phosphate buffer, pH 6.2 (5), was added. The sample was then blendedusing a Stomacher (Model 400, Cooke Laboratory Products, Alexandria,Va.) for 2 min. After blending, the contents of the bag were filteredthrough cheesecloth. The mixture was centrifuged at 5000×g for 10 min.and the aqueous supernatant fraction was analyzed for the presence oftoxin. The extraction procedure allows complete recovery of added toxin.For toxin assays of TPYG broth employed in the MPN procedure, the mediumwas centrifuged at 5000×g for 10 min., and the supernatant was analyzedfor the presence of toxin.

In the mouse protection assay for botulinal toxin (see FDABacteriological Analytical Manual for Foods, 1976), each of two mice wasinnoculated intraperitoneally with 0.5 ml of the extract from the testsample. Mice were held for up to four days and examined for symptoms anddeath characteristic of C. botulinum intoxication. When death occurred,two additional mice were challenged with a sample-antitoxin mixturewhich was pre-incubated at 37° C. for 30 min. Unprotected controls wereagain inoculated so that they could be tested simultaneously with theprotected mice. Nonspecific deaths due to the meat extracts did notoccur.

Chicken frankfurters: A standard chicken frankfurter emulsion wasobtained from ABC Research Laboratories. This emulsion consisted ofmechanically deboned chicken meat, 550 ppm sodium erythorbate, 2.0%dextrose, 0.25% white pepper, 0.07% nutmeg, and 0.5% liquid smoke. Saltat 2.5% was added during the inoculation phase. The proximatecomposition of the emulsion was approximately 12% protein, 20% fat, and67% moisture.

Formulations for chicken frankfurter emulsions: Chicken frankfurteremulsions were prepared at 5 different nisin levles: 0, 12.5 ppm, 100ppm, 250 ppm and 500 ppm. Three different nitrite levels will also beemployed: 0, 40ppm, 80 ppm and 120 ppm. Previous experience hasindicated that emulsions made with these levels of nitrite and noadditional antibotulinal agents will support the outgrowth and toxinproduction by C. botulinum. In addition, a control batch containing 156ppm nitrite and no nisin was prepared. Chicken frankfurter emulsionsmade with 156 ppm nitrite should not support botulinal outgrowth sincethis is the level of nitrite added commercially. A total of 22 differentformulations with different combinations of nitrite and nisin wereevaluated. Some batches were prepared on two separate occasions so thata portion of the study could be repeated.

Preparation and inoculation of chicken frankfurther emulsions: With amixer, the prepared chicken frankfurter emulsion, the heat-shocked (80°C., 10 min.) spore inoculum, sodium nitrite and/or nisin, and salt weremixed. The salt was added last to avoid extraction of salt-solubleproteins which would result in a sticky emulsion with poor mixingproperties. The spores and antibotulinal agents were well mixed beforeaddition of the salt. The final spore load approximated 1000 spores pergram of emulsion. After mixing the emulsion was placed in 18×150 mm testtubes. The tubes were heat processed in a forced air oven. The productwas slowly cooked to a final internal temperature of approximately 71°C. over approximately a 2 hour period. After cooking, the tubes werecooled, capped with vaspar, and incubated as described below.

Incubation and sampling schedule for chicken frankfurter emulsions:Samples were incubated in two different ways. In the first method, tubeswere held at 27° C. for up to 6 weeks. In the second method, tubes wereheld at 4° C. for 8 weeks followed by incubation at 27° for up to 6weeks. The second method was invoked to study the effects of nisindecomposition in chicken frankfurters during refrigerated storage andany resultant impact on the antibotulinal effectiveness of nisin. Thedecreased effectiveness of nisin in cooked meat medium was attributed toits binding to meat particles (Scott and Taylor, noted above). The slowdecomposition of nisin in food systems has been observed previously.These factors may decrease the effectiveness of nisin in meat systems.The 27° C. incubation temperature was chosen because this abusetemperature has been widely used in previous studies on botulinaloutgrowth in meat systems.

Each experimental batch was examined for spoilage (gas, uneven color,phase separation, liquefaction) at weekly intervals following incubationat 27° C. Each week, 7 samples were removed: 5 for toxin analysis and 2for nisin analysis. Tubes with evidence of spoilage were preferentiallyselected. The tubes selected for refrigerated storage were sampled fornisin levels at 0, 4, and 8 weeks of refrigerated storage. Aftertransfer to 27° C. incubation, the sampling schedule was identical tothat given above.

Each batch of the chicken frankfurter emulsion was analzyzed forresidual nitrite, number of viable C. botulinum organisms, fat,moisture, and pH after mixing and preparation were complete but prior toincubation.

Chemical analyses: The pH of the chicken frankfurter emulsion wasdetermined by placing the electrode directly in the mixture. Fat,moisture, NaCl, and residual nitrite were determined by standard AOACmethods. Nisin analysis was performed by the Micrococcus flavus assaymethod of Tramer and Fowler, J. Sci. Food Agric. 15:522-528.

Results and Discussion. The formulations used in the various batches arereported in Table 4. Twenty-two different batches were used. Two batcheswere prepared at 12.5 ppm nisin. When the batch with 12.5 ppm nisin and80 or 120 ppm nitrite did not prevent toxin formtion, it was obviousthat batches prepared at 12.5 ppm nisin and either 0 or 40 ppm nitritewould not yields valuable information. A similar logic was used toeliminate the batch containing 100 pm nisin and 40 ppm nitrite. Batcheswere added that contained 500 ppm nisin and either 0 or 40 ppm nitritein an attempt to determine if such high nisin levels would providecomplete protection.

Each batch was analyzed for moisture content, fat content, and pHimmediately after formulation. The data is included in Table 4. Themoisture levels ranged from 58.5% to 66.9% which was slightly below theoriginal goal of 67% moisture. Some moisture was probably lost duringthe heating process. The fat content ranged from 18.6% to 24.7% whichconformed closely to the goal of 20% fat. The pH range for the variousbatches was from pH 5.5 to pH 6.2 with all but one batch falling in therange of pH 5.9 to pH 6.2. The actual spore loads obtained in each batchof emulsion were estimated following formulation (Table 4). Spore loadsranged from 220 to 2600 spores/g but 9 of the 13 batches were in therange of 800 to 2300 spores/g. Spore counts are notoriously inaccurateso these counts should be considered as estimates. The internaltemperature achieved with each batch of emulsion in the heating processis also given in Table 4. The final internal temperatures ranged from66° C. to 74° C.

                  TABLE 4                                                         ______________________________________                                        Analysis of Chicken Frankfurter Formulations                                                                             Final                                                                  Spore  In-                                                   Mois-            Count  ternal                             Batch              ture    Fat      (spores/                                                                             Temp.                              No.   Formulation  (%)     (%)  pH  g)     (° C.)                      ______________________________________                                        1     500    ppm nisin 63.6  20.0 5.9  800   71                                     0      ppm nitrite                                                      2     500    ppm nisin 58.8  18.9 5.9 1500   72                                     40     ppm nitrite                                                      3     250    ppm nisin 64.4  22.1 5.5  270   73                                     0      ppm nitrite                                                      4     250    ppm nisin 61.8  20.0 6.0 1700   72                                     40     ppm nitrite                                                      5     250    ppm nisin 64.3  18.6 6.0 1200   68                                     120    ppm nitrite                                                      6     250    ppm nisin 63.1  20.2 5.9 1500   69                                     250    ppm nitrite                                                      7     100    ppm nisin 63.3  18.6 6.1 2600   68                                     0      ppm nitrite                                                      8     100    ppm nisin 65.4  18.6 6.1  220   74                                     120    ppm nitrite                                                      9     12.5   ppm nisin 63.5  18.8 6.1 1700   72                                     120    ppm nitrite                                                      10    0      ppm nisin 61.8  21.7 6.0 1700   71                                     0      ppm nitrite                                                      11    0      ppm nisin 63.5  24.1 6.0 1800   72                                     40     ppm nitrite                                                      12    0      ppm nisin 66.9  24.7 6.0  220   72                                     120    ppm nitrite                                                      13    0      ppm nisin --    --   --  2300   66                                     156    ppm nitrite                                                      ______________________________________                                    

Several batches of frankfurter emulsion were formulated a second time sothat a portion of the study could be repeated. The data on added levelsof nisin and nitrite, % moisture, % fat, pH, spore count, and finalinternal temperature for these batches is provided in Table 5. For thesebatches, a separate lot of frankfurter emulsion prepared to identicalspecifications was obtained from ABC Research Laboratories. The data inTable 5 indicate that the moisture levels obtained in these batches werea little higher, and the fat content was a little lower. Temperaturecontrol was improved because a different and better oven was used.

                  TABLE 5                                                         ______________________________________                                                                                   Final                                                                  Spore  In-                                                   Mois-            Count  ternal                             Batch              ture    Fat      (spores/                                                                             Temp.                              No.   Formulation  (%)     (%)  pH  g)     (° C.)                      ______________________________________                                        14    0      ppm nisin 69.0  13.4  6.14                                                                             1100   71                                     156    ppm nitrite                                                      15    0      ppm nisin 67.7  --   6.0 1050     72.5                                 120    ppm nitrite                                                      16    12.5   ppm nisin 69.4  --   6.1 1100   71                                     120    ppm nitrite                                                      17    100    ppm nisin 67.0  --   6.0 3000   71                                     120    ppm nitrite                                                      18    250    ppm nisin 64.9  --   6.1 5000   71                                     120    ppm nitrite                                                      19    0      ppm nisin 58.5  17.8 6.2 2200   71                                     80     ppm nitrite                                                      20    12.5   ppm nisin 66.1  11.8 6.1 2000   71                                     80     ppm nitrite                                                      21    100    ppm nisin 59.0  15.4 6.1 1100   69                                     80     ppm nitrite                                                      22    250    ppm nisin 63.4  16.8 5.9 1700   69                                     80     ppm nitrite                                                      ______________________________________                                    

The results of the analysis for botulinal toxin following incubation wasshown in Tables 6 and 7. The results from the 27° C. incubation areshown in Table 6. Table 7 gives the results obtained with 8 weeks ofrefrigerated storage followed by incubation at 27° C. As mentionedpreviously, some batches were repeated. The results are recordedseparately on Tables 6 and 7.

                                      TABLE 6                                     __________________________________________________________________________    Botulinal Toxin Formation in Chicken Frankfurter Emulsions Incubated at       27° C.                                                                  Number of toxic samples/Number of samples tested                             ppm nisin                                                                     500     500                                                                              250                                                                              250                                                                              250                                                                              250                                                                              250 100                                                                              100                                                                              100                                                                              12.5                                                                              12.5                                                                             0  0  0   0  0   0                 ppm nitrite                                                                   Week                                                                              40  0  250                                                                              120                                                                              80 40 0   120                                                                              80 0  120 80 156                                                                              156                                                                              120 40 80  0                 __________________________________________________________________________    0   0/3 0/3                                                                              0/3                                                                              0/3                                                                              0/2                                                                              0/3                                                                              0/3 0/3                                                                              0/2                                                                              0/3                                                                              0/3 0/2                                                                              0/2                                                                              0/2                                                                              0/3 0/3                                                                              0/2 0/3               1   1/5 0/5                                                                              0/5                                                                              0/5                                                                              0/5                                                                              5/5                                                                              5/5 0/5                                                                              0/5                                                                              0/5                                                                              2/5 4/5                                                                              0/10                                                                             0/5                                                                              5/5 5/5                                                                              5/5 5/5               2   2/5 5/5                                                                              0/5                                                                              0/5                                                                              1/5                                                                              -- --  0/5                                                                              3/5                                                                              5/5                                                                              1/5 5/5                                                                              0/10                                                                             1/5                                                                              --  --     --                3   1/5 5/5                                                                              0/5                                                                              0/5                                                                              4/5                                                                              -- --  0/5                                                                              5/5                                                                              -- 1/5    -- 0/5                                                                              --  --     --                4   1/5 -- 0/5                                                                              0/5                                                                              5/5                                                                              -- --  0/5   -- 0/5    --  9/10                                                                            --  --     --                5   4/5 -- 0/5                                                                              0/5   -- --  1/5   -- 7/7    -- -- --  --     --                6   4/4 -- 0/5                                                                              1/5   -- --  6/6   -- --     -- -- --  --     --                __________________________________________________________________________

                                      TABLE 7                                     __________________________________________________________________________    Botulinal Toxin Formation In Chicken Frankfurter Emulsion Incubated In        The Refrigerator For 8 Weeks And Then At 27° C.                         Number of toxic samples/Number of samples tested                             ppm nisin                                                                     500    500                                                                              250                                                                              250                                                                              250 250                                                                              250                                                                              100                                                                              100 100                                                                              12.5                                                                             12.5                                                                              0  0   0  0  0  0                  ppm nitrite                                                                   Week                                                                              40 0  250                                                                              120                                                                              120 40 0  120                                                                              120 0  120                                                                              120 156                                                                              156 120                                                                              120                                                                              40 0                  __________________________________________________________________________    0.sup.a                                                                           0/2                                                                              0/2                                                                              0/2                                                                              0/2                                                                              0/2 0/2                                                                              0/2                                                                              0/2                                                                              0/2 0/2                                                                              0/2                                                                              0/2 -- 0/2 0/2                                                                              0/2                                                                              0/2                                                                              0/2                4 days.sup.a                                                                      -- -- -- -- --  -- 0/5                                                                              -- --  2/5                                                                              -- --  -- --  2/5                                                                              -- 5/5                                                                              0/5                1.sup.a                                                                           0/5                                                                              0/5                                                                              0/5                                                                              2/5                                                                              1/5 5/5                                                                              5/5                                                                              2/5                                                                              4/5 5/5                                                                              4/5                                                                              10/10                                                                             -- 4/5 5/5                                                                              5/5                                                                              5/5                                                                              5/5                2.sup.a                                                                           4/5                                                                              2/5                                                                              0/5                                                                              3/5                                                                              10/10                                                                             -- -- 5/5                                                                              10/10                                                                             -- 5/5                                                                              --  -- 10/10                                                                             -- -- -- --                 3.sup.a                                                                           5/5                                                                              5/5                                                                              5/5                                                                              5/5                                                                              --  -- -- -- --  -- -- --  -- --  -- -- -- --                 __________________________________________________________________________     .sup.a Incubation period at 27° C.                                

Nisin alone, even at 500 ppm, did not prevent botulinal outgrowth andtoxin production in chicken frankfurter emulsion. Nisin at 500 ppm diddelay toxin production by one week. At lower nisin concentrations in theabsence of nitrite, toxin was present in all tubes by the end of oneweek of incubation at 27° C. Likewise, nitrite alone at levels up to 120ppm did not prevent botulinal outgrowth and toxin production. All tubeswere positive for toxin by the end of the first week of incubation at27° C. This lack of botulinal inhibition at levels of 120 ppm or less ofnitrite was expected based on earlier work in chicken frankfurteremulsions by Sofos et al, J. Food Sci. 44:668-672 (1973). The negativecontrol containing no nisin and 156 ppm nitrite was not expected toallow toxin production in the first few weeks of incubation. In thefirst trial, no toxin was found in the emulsion containing 156 ppmnitrite after two weeks of incubation.

Unforunately, the incubation of the negative control had to bediscontinued at this point due to heavy bacterial growth and gasproduction. The bacteria growing in the 156 ppm emulsion were not C.botulinum but some unidentified bacteria, perhaps lactics. Thisnon-botulinal growth was only observed with this particular batch ofemulsion. Other batches that showed signs of bacterial growth also hadtoxin indicating that at least some of the growth was C. botulinum. Thisproblem with the 156 ppm nitrite control was eliminated in the secondtrial probably due to improved temperature control during cooking. The156 ppm control batch did not become toxic until the fourth week in thissecond experiment (Table 6).

Nisin and nitrite worked together to prevent or delay botulinal toxinproduction in chicken frankfurter emulsions. In batches containing 120ppm nitrite and either 100 or 250 ppm nisin, toxin formation was delayeduntil the fifth or sixth week of incubation at 27° C. Intermediateeffects were noted with combinations of 80 ppm nitrite with 100 or 250ppm nisin. This is an improvement over the protection provided by 156ppm nitrite alone (Table 6). Toxin formation was totally prevented inthe batch prepared with 250 ppm nisin and 250 ppm nitrite but, since nocontrol was available with 250 ppm nitrite alone, no conclusions can bereached regarding the role of nisin in this result. Toxin formation wasslowed and sporadic in batches containing 12.5 ppm nisin and 120 ppmnitrite or 500 ppm nisin and 40 ppm nitrite.

These studies demonstrate the effectiveness of using nisin atappropriate levels suited to the product in question to control thegrowth of C. botulinum spores.

What is claimed is:
 1. A process for controlling the growth ofClostridium botulinum spores and the elaboration of botulinum toxintherefrom in a food product excluding a high moisture contentpasteurized process cheese product, said process comprising adding tosaid product an amount of nisin or a nisin-producing culture sufficientto inhibit botulinum spore growth.
 2. The process according to claim 1in which from about 2,000 to about 10,000 International Units of nisinare added per gram of food product.
 3. A food product containing fromabout 100 to about 300 parts per million nisin produced by the processof claim
 1. 4. The process as claimed in claim 1 in which an amount ofat least 2000 Internation Units (50 ppm) of nisin is added orincorporated per gram of food product.
 5. The process as claimed inclaim 4 in which an amount of at least 4000 International Units (100ppm) of nisin is added or incorporated per gram of food product.
 6. Aprocess as claimed in claim 4 in which an amount up to 12,000International Units (300 ppm) of nisin is added or incorporated per gramof food product.
 7. A food product excluding a high moisture contentpasteurized process cheese product, said food product containing anamount of nisin or a nisin-producing culture sufficient to inhibitbotulinum spore growth.
 8. The food product as claimed in claim 7containing at least 2,000 IU (50 ppm) of nisin.
 9. A process forcontrolling the growth of Clostridium botulinum spores and theelaboration of botulinum toxin therefrom in a food product selected fromthe group consisting of tomato products, cold meat products and wet fishsystems, said process comprising adding to said food product from about2,000 to about 10,000 International Units of nisin or a nisin-producingculture per gram to inhibit botulinum spore growth.
 10. The process asclaimed in claim 9 in which an amount of at least 2,000 InternationalUnits (50 ppm) of nisin is added or incorporated per gram of foodproduct.
 11. The process as claimed in claim 10 in which an amount of atleast 4,000 International Units (100 ppm) of nisin is added orincorporated per gram of food product.
 12. A process as claimed in claim10 in which an amount up to 12,000 International Units (300 ppm) ofnisin is added or incorporated per gram of food product.
 13. The processas claimed in claim 9 in which the food product is a tomato product. 14.The process as claimed in claim 9 in which the food product is a wetfish system.
 15. The process as claimed in claim 9 in which the foodproduct is a cold meat product.
 16. The process as claimed in claim 15in which the product is chicken frankfurter emulsion.